Plastids of higher plants, i.e. chloroplasts, amyloplasts and chromoplasts, have the same genetic content, and thus are believed to be derived from a common precursor, known as a proplastid. The plastid genome is circular and varies in size among plant species from about 120 to about 217 kilobase pairs (kb). The genome typically includes a large inverted repeat, which can contain up to about 76 kilobase pairs, but which is more typically in the range of about 20 to about 30 kilobase pairs. The inverted repeat present in the plastid genome of various organisms has been described (Palmer, J. D. (1990) Trends Genet. 6:115-120).
One advantage of plant plastid transformation over nuclear transformation is that the plastids of most plants are maternally inherited, and consequently heterologous plastid genes are not pollen disseminated. This feature is particularly attractive for transgenic plants having altered agronomic traits, as introduced resistance or tolerance to natural or chemical conditions will not be transmitted to wild-type relatives.
Plant plastids are also major biosynthetic centers. In addition to photosynthesis in chloroplasts, plastids are responsible for production of important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments.
Plastids can be present in a plant cell at a very high copy number, with up to 50,000 copies per cell present for the chloroplast genome (Bendich, A. J. (1987) BioEssays 6:279-282). Thus, through plastid transformation plant cells can be engineered to maintain an introduced gene of interest at a very high copy number.
For all of the above reasons, the plastids of higher plants present an attractive target for genetic engineering. Stable transformation of plastids has been reported in the green algae Chlamydomonas (Boynton et al. (1988) Science 240:1534-1538) and more recently in higher plants (Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530: Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917); (Staub, J. M. and Maliga, P. (1993), EMBO J. 12:601-606). The method disclosed for plastid transformation in higher plants relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination.
There is a continuing need to introduce newly discovered or alternative Bacillus thuringiensis genes into crop plants. Cry proteins (d-endotoxins) from Bacillus thuringiensis have potent insecticidal activity against a number of Lepidopteran, Dipteran, and Coleopteran insects. These proteins are classified CryI to CryV, based on amino acid sequence homology and insecticidal activity. Most CryI proteins are synthesized as protoxins (ca. 130-140 kDa) then solubilized and proteolytically processed into active toxin fragments (ca. 60-70 kDa).
The poor expression of the protoxin genes from the nucleus of plants has heretofore required the use of `truncated` versions of these genes. The truncated versions code only for the active toxin fragments. Other attempts to increase the expression efficiency have included resynthesizing the Bacillus thuringiensis toxin genes to utilize plant preferred codons. Many problems can arise in such extensive reconstruction of these large cry genes (approximately 3.5 Kb), and the process is both laborious and expensive.
Problems can also arise as new insect pests become endemic, or as existing populations develop resistance to a particular level or type of Bacillus thuringiensis toxin. Thus, there is a particular need for producing higher and thereby more effective levels of the Bacillus thuringiensis toxin in plants, a need which will only increase with time.